PROJECT SUMMARY/ABSTRACT Stereocilia are the sensory machinery of the inner ear sensory hair cells. We found that the stability of these actin-filled projections depends on the constant influx of calcium through the mechano-electrical transduction (MET) channels that are located at the tips of stereocilia (Velez-Ortega, et al., eLife, 2017). We found that the blockage of MET channels or the breakage of their gating tip links leads to the shortening of stereocilia; but once the blockage is removed or the tip links regenerate, stereocilia are able to regrow. Based on the changes in stereocilia height in response to variations in the resting MET current, I hypothesized that the stereocilia actin cytoskeleton exhibits activity-dependent plasticity. Aim 2 of the parent grant (R21 DC017247) has been studying the contribution of myosin XV isoforms in the MET-dependent regulation of stereocilia height using several myosin XV mutant mice. Our results thus far indicate that the two types of mammalian auditory hair cells (inner vs. outer hair cells) have different molecular mechanisms driving the MET-dependent changes in the stereocilia cytoskeleton. In addition, we have observed interesting MET-dependent changes to the resting tilt of the hair bundle that could arise from changes at the level of the stereocilia rootlets and/or the cuticular plate. The goal of this Administrative Supplement is to obtain resources (for personnel and supplies) that would allow us to perform focused ion beam scanning electron microscopy (FIB-SEM) imaging to (i) answer some questions that traditional SEM imaging has not been able to address due to the difficulty in obtaining accurate 3D measurements of the extremely short stereocilia in some myosin XV mutants; and, using the same FIB-SEM data sets, to (ii) further explore the mechanotransduction-dependent rearrangements of the stereocilia rootlets and/or cuticular plate. In addition, this supplement would allow Dr. Velez-Ortega to spend less time on the bench performing experiments and, instead, more time on data analysis and manuscript preparation. This `shift' in the focus of Dr. Velez-Ortega's effort would minimize a decrease in the progress of the parent grant due to the upcoming birth of her child in the Fall of 2020.